Leukocyte CH25H is a potential diagnostic and prognostic marker for lung adenocarcinoma

Metastasis, a major challenge during the treatment of lung cancer, causes deterioration in patient health outcomes. Thus, to address this problem, this study aimed to explore the role and contribution of Cholesterol 25-Hydroxylase (CH25H) as a potential diagnostic and prognostic marker in lung cancer. Online public databases were used to analyze the expression level, prognostic value, gene-pathway enrichment, and immune infiltration of CH25H in lung cancer patients. The Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) was used to analyze and detect the CH25H expression levels in leukocytes from lung cancer patients. The expression level of CH25H was significantly reduced in lung adenocarcinoma (LUAD), which is associated with a higher disease stage, but not in lung squamous cell carcinoma (LUSC). Kaplan–Meier survival analysis indicated that LUAD patients with low CH25H expression had a worse prognosis. Mechanistically, our results showed that in LUAD, CH25H may be a regulatory factor affecting the immune cell infiltration level, and the resultant tumor development. Experimental data showed that low expression of CH25H in leukocytes was significantly associated with LUAD metastasis (P < 0.01). Our study suggests that CH25H may function as a prognostic and risk stratification biomarker for LUAD.

Immune infiltration analysis. Sangerbox Tools (http:// www. sange rbox. com/ tool) are a free online platform for comprehensive data analysis. We used Sangerbox Tools to estimate the correlation between CH25H expression and immune infiltration level in LC patients according to previous literature 16 and combined it with data from TCGA for prognosis and immunotherapy analysis.
Ethics approval and consent to participate. This study was conducted according to the principles of the World Medical Association Declaration of Helsinki and approved by the ethics committee of the second Hospital of Jiaxing (IRB approval No. JYEY2017023). All participants provided written informed consent for this study. Other datasets were retrieved from the published literature, so it was confirmed that all written informed consent was obtained.
Patient selection and evaluation. The patients included in this study had primary LC treated at the Second Hospital of Jiaxing (Zhejiang, China) from June 2019 to August 2020. The cancer was diagnosed by clinical and X-ray findings and confirmed via histology of tumor biopsies. TNM and final staging was performed as per the American Joint Committee on Cancer guidelines (AJCC) 8th edition. The criteria to judge whether the tumor is metastatic is whether the tumor have invaded bone, brain and other distant organ or lymph nodes, also identified as M1 in AJCC staging system. The inclusion criteria included previously employed surgery and/ or standardized treatment and histological confirmation of non-small cell lung cancer (NSCLC). The exclusion criteria included multiple organ dysfunction or a history of organ transplantation, small cell lung cancer (SCLC), unwillingness to participate in the study, and cessation of therapy. Data regarding the clinical characteristics were carefully collected.
Determination of CH25H mRNA expression. The blood specimens (10 mL) from all the enrolled patients, including 15 healthy volunteers (non-cancer individuals) were collected and centrifuged at 2500 rpm for 10 min within 2 h of their collection to separate the leukocytes. Total RNA extraction from leukocytes was performed using an RNA Plus Kit (Yunying, Jiaxing, China). Reverse transcription was performed using the HiScript® III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China), and qRT-PCR analysis was performed using the Alldetect™ kit (Yunying, Jiaxing, China) on an ABI 7500 system (ThermoFisher, MA, USA). The TaqMan assay was used for qRT-PCR amplification detection. Ribosomal protein LO (RPLO) was used as the reference gene, and primers and probes (Table S1) were designed using Primer Premier 5.0 (Premier Biosoft, CA, USA), and synthesized by General Biotech (Shanghai, China). Each process was strictly performed according to the manufacturer's protocol.

Statistical analyses.
The expression level of CH25H across different LC groups was analyzed by the Mann-Whitney U test. Statistical analyses were performed with SPSS v.22.0 (IBM Corp., New York, USA). Statistical graphs were generated by the R package "ggplot2". A P < 0.05 was considered statistically significant and was used as the inclusion standard in the analysis.

Results
CH25H expression level downregulated in LUAD and correlated with prognosis. We analyzed the expression level of CH25H in lung tumor tissues and normal tissues using online public databases (Fig. 1A). The CH25H expression levels showed highly significant differences (p < 0.001), the CH25H mRNA expression level in the metastatic group was significantly down-regulated in LUAD compared to the non-metastatic group www.nature.com/scientificreports/ ( Fig. 1B). Compared with normal tissues, CH25H expression was significantly down-regulated in the metastatic group (p < 0.001), however, no significant difference was found between the non-metastatic group in LUAD (Fig. 1B). In LUSC, we found that the expression level of CH25H was significantly lower than that in normal tissues in both metastatic and non-metastatic groups, but no significant difference was found between the metastatic and non-metastatic groups (Fig. 1C). We observed that in LUAD, this negative expression was significantly associated with sex ( Fig. S1B), and nodal metastasis status (Fig. S1F). Other factors such as age (Fig. S1A), race ( Fig. S1C), smoking habits (Fig. S1D), and histological subtypes (Fig. S1E) showed poor correlation with CH25H expression. However, in patients with LUSC, no outstanding relationship between CH25H expression and these factors was observed (Fig. S2). The survival difference was evaluated, and results showed that compared with the low expression cohort, patients in the high expression cohort showed a significantly enhanced OS (P < 0.01, N = 478), indicating a better prognosis (Fig. 1D). However, compared with the lower expression cohort, LUSC patients with high CH25H expression showed a reduced trend of survival time in both OS, although the difference was not statistically significant (P > 0.05, N = 481, Fig. 1E).
CH25H associated with immune regulation. Heat maps showed that the top 20 significant genes were positively and negatively related to CH25H in LUAD ( Fig. 2A,B) and LUSC (Fig. 2E,F), respectively. Notably, 6/20 genes (CD1C, CD74, HLA-DPB1, NLRP3, ADRB2 and AMICA1) were positively related to CH25H in LUAD. They were therefore responsible for the positive regulation of the immune system [17][18][19][20][21][22] . Pathway analysis revealed an enriched immune regulation, such as the regulation of leukocyte activation, positive regulation of immune response, and adaptive immune system (Fig. 2C,D). However, in LUSC, CH25H showed a weak association with the immune regulation, as analyzed by gene enrichment (Fig. 2E,F) and pathway analysis (Fig. 2G,H). These results could possibly explain the importance of CH25H as a prognostic biomarker in case of LUAD only. It was therefore hypothesized that CH25H controls the spread and infiltration of tumor cells via immune regulation in LUAD.
Some studies have found that biomarkers such as CLEC3B 26 and YTHDF1/YTHDF2 27 affect the immune microenvironment, and metastasis and prognosis of lung cancer. Therefore, we studied the correlation between CH25H expression and immune cell infiltration.
CH25H expression level associated with immune infiltration level. We hypothesized that CH25H expression regulates the infiltration levels of immune cells. We also investigated the relationship between CH25H expression and the abundance of immune infiltrates. The results showed that CH25H expression level was less associated with the infiltration of immune cells (for instance, B cells, T cells, and macrophages) in the LUSC subtype. However, the expression of CH25H was significantly positively correlated with immune cell infiltration in LUAD (Fig. 3A). These results confirmed that CH25H expression potentially regulates the immune infiltration in patients with LUAD.
In addition, we observed that CH25H expression was significantly correlated with immune infiltration in LUAD (Fig. 3B,D), better than in LUSC (Fig. 3E-G), as evaluated by stromal, immune, and comprehensive ESTIMAT, respectively.
Immune infiltration level associated with prognosis in LUAD. We further investigated the relationship between immune score and OS of LC patients, and the results (Fig. 4A,B) were similar to the result www.nature.com/scientificreports/ of CH25H expressional level and OS (Fig. 1D,E). The survival difference results showed that, compared with the low immune score cohort, the high immune score cohort showed a significantly enhanced OS (P < 0.05) in LUAD (Fig. 4A). Similar analyses were performed for LUSC, but the opposite result was obtained (P > 0.05, Fig. 4B).
CH25H level associated with immunotherapy in LUAD. Immunotherapy molecular marker analysis revealed that both, CD274 (P < 0.001) and CTLA4 (P < 0.01) significantly correlated with CH25H expression in LUAD (Fig. 4C,D), this was not seen in the case of PDCD1 (Fig. S3 (left)). However, in LUSC, only CD274 was significantly related to CH25H expression (Fig. 4E) but not CTLA4 (Fig. 4F) or PDCD1 (Fig. S3 (right)). The results of this study indicate that CH25H can be a potential biomarker for predicting immunotherapy efficacy.
Leukocytes CH25H expression level related to the lung tumor metastasis. To confirm the role of CH25H in lung cancer, we conducted a preliminary clinical study using leukocyte samples from patients having lung cancer and non-cancer individuals. This study employed 34 patients newly diagnosed with LC between 2019 and 2020. The average age at the time of diagnosis was 64 years and the median age of the cohort was 65 years (range 38-88 years). Other characteristics of the study participants are listed in Table 1.
Based on the tumor metastasis status, the patients were divided into metastatic, non-metastatic and normal groups. Blood samples were collected to detect CH25H mRNA expression levels in leukocytes. The results showed that, in LUAD patients, CH25H expression was significantly decreased in the metastatic group (P < 0.01, Fig. 5A). However, no significant decrease in the CH25H expression was observed in LUSC patients with tumor metastasis (P > 0.05, Fig. 5B), indicating that the CH25H expression level is a potential biomarker for LC metastasis prediction, especially in LUAD. Additionally, compared with the normal group, the expression level of CH25H in the metastatic group was significantly decreased (P < 0.01), but no significant difference was found in the non-metastatic group, either the LUAD or LUSC dataset. www.nature.com/scientificreports/

Discussion
Previous research has shown that low CH25H levels in leukocytes correlate with poor prognosis in melanoma patients 9 . Mechanistically, CH25H catalyzes cholesterol oxidation, wherein cholesterol is metabolized to 25-hydroxycholesterol (25-HC) in vivo, which could resist the normal cell absorption of the TEVs (tumorderived extracellular vesicles), indicating the important role of CH25H in defense against the production of the tumor pre-metastatic microenvironment. In this study, we found that low CH25H levels were correlated with poor prognosis in patients with LUAD (Fig. 1D). Genes co-expressed with CH25H were analyzed to understand the underlying mechanism of CH25H in LUAD. We found that some genes that were positively related to CH25H were related to immunity (CD1C, CD74, HLA-DPB1, NLRP3, ADRB2 and AMICA1) ( Fig. 2A). According to Di Blasio et al., CD1C (+) dendritic cells (DCs) can elicit immunogenic cell death (ICD) 17 . Overexpression of HLA-DPB2 promotes tumor immune infiltration by regulating HLA-DPB1, which is associated with a high survival rate in breast cancer 18 . Lu et al. showed that NLRP3 inflammasome blockade down-regulates the expression of PD-L1 and reduces the immunosuppression of lymphoma 19 . The expression of ADRB2 is positively correlated with the infiltration of immune cells in breast cancer, especially the T cells 20 . Activated cGAS-STING signaling in LUAD by AMICA1 induces immune cell infiltration 21 . High infiltration of CD74 + macrophages in hepatocellular cancer is related to higher infiltration of CD8 + cytotoxic T cells (CTL) 22 . These previous studies suggest that CH25H may also be associated with positive regulation of tumor immunity in LUAD. Further, pathway analysis showed that CH25H may participate in several immune-related processes, including regulation of leukocyte activation, positive regulation, immune response, and adaptive immune system (Fig. 2C,D), which confirmed our hypothesis.
In our study, according to the Sangerbox tool, we found that CH25H is positively related to the immune score and the infiltration of several immune cells, including B cells, CD4 + T cells, CD8 + T cells, dendritic cells, macrophages, and neutrophils (Fig. 3A), which is consistent with the role of AMICA1 in LUAD according to a previous study 21 . This means that CH25H may also affect the level of immune cell infiltration through signaling pathways such as cGAS-STING, however, further research is required. The infiltration level of immune cells is closely related to the effectiveness of immunotherapy and classification basis of "hot" (highly infiltrated) and www.nature.com/scientificreports/  www.nature.com/scientificreports/ "cold" (non-infiltrated) tumors 23 . High CH25H expression may cause high infiltration level, i.e. "hot" tumor. Furthermore, they may influence the immunotherapy, manifested by an upregulation of the level of immunotherapy molecular markers (such as CD274 and CTLA4, Fig. 4, left plot) in LUAD. Moreover, CD8 + T cells, one of the most important effector cells, play important roles in clearing intracellular pathogens and tumors 23 . So, mechanistically, these results indicated that CH25H may affect tumor progression via immune regulation in LUAD. This may also explain the reason behind the decreased CH25H expression levels with the progression of LUAD (Fig. 1B). Moreover, leukocyte CH25H expression analysis revealed that CH25H expression levels were significantly down-regulated in the metastatic cohort. The main product of CH25H catalyzed reaction is 25-HC, which has been reported to suppress the migration and proliferation of gastric cancer cells 24,25 , and the opposite effect was observed in LUAD 26 . Our results are consistent with the outcomes of these studies; when the donor suffered from LUAD, decreased CH25H expression reduced the 25-HC level, thereby possibly promoting tumor metastasis, as shown in Fig. 5. Our hypothesis may explain this phenomenon. Decreased CH25H expression in LUAD may cause low immune cell infiltration, causing tumor cells to escape immune system and promote tumor metastasis, finally leading to poor prognosis, as shown in Figs. 1D and 4A.
However, this study had some limitations. First, further studies consisting of cell experiments and clinical research are required to validate and explore the potential molecular mechanisms underlying the correlation between CH25H expression and immune response. In addition, this was a single-center study with a small sample size. Therefore, prospective multicenter studies involving a larger cohort should be undertaken to expand our understanding of the ability of leukocyte CH25H expression to distinguish between metastatic and nonmetastatic LC patients. Most importantly, tumor tissue experiments are needed to verify the accuracy of leukemia results.

Conclusion
Here, through bioinformatics analysis, we speculate that CH25H expression levels are associated with immune cell infiltration. This was confirmed by the detection of CH25H expression in leukocytes. We identified a potential molecular marker for the prognosis and risk stratification of LUAD.

Clinical practice points.
Previous studies have indicated that the expression level of CH25H is an independent prognostic factor for predicting distant metastasis of breast cancer and melanoma, however, its prognostic value in lung cancer remains unclear. Our data showed a decreased CH25H expression in LUAD. Notably, low expression of CH25H in leukocytes is an obvious molecular characteristic of metastatic tumors which results in poor prognosis. In addition, increased CH25H expression improves the overall survival time of patients with LUAD, displaying a potential correlation with immune infiltration. Our findings promote an improved understanding of the mechanisms of CH25H in lung cancer and demonstrate that leukocyte CH25H expression could be used to develop a rapid, inexpensive, and powerful diagnostic strategy for predicting the risk of lung cancer metastasis. This study will also serve as a foundation for future large-cohort studies to verify the significance of CH25H expression.

Figure 5.
Correlations between lung tumor metastasis and CH25H expression. (A) Box plot showed that significant difference (p < 0.001) in CH25H expression between metastatic and non-metastatic LUAD patients, and significant difference (p < 0.001) were also found between metastatic group and normal group; (B) significant difference were only found between metastatic LUSC patients and normal individuals. "***" and "NS" indicate p < 0.001 and p > 0.05, respectively. www.nature.com/scientificreports/

Data availability
The data that supporting the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.